Ollivier Milhavet

Successful Transmission of Three Mouse-Adapted Scrapie Strains to Murine Neuroblastoma Cell Lines Overexpressing Wild-Type Mouse Prion Protein

ABSTRACT Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrPC-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrPC than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrPSc, in the agent-inoculated PrP-overexpressing cells, while no PrPSc was detectable in the untransfected cells after 3 passages. Production of PrPSc in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrPSc-A strains Chandler, 139A, and 22L but not to PrPSc-B strains 87V and 22A. We further demonstrate the successful transmission of PrPSc from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrPC and the sequence of PrPSc. This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.

Oxidative stress and the prion protein in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic or genetic in origin. These diseases are believed to be the consequence of the conformational conversion of the prion protein into an abnormal isoform. Their exact pathogenic mechanism remains uncertain, but it is believed that oxidative stress plays a central role. In this article, we will first review in detail the data supporting the latter hypothesis. Subsequently, we will discuss the relationship between the prion protein and the cellular response to oxidative stress, attempting ultimately to link PrP function and neurodegeneration in these disorders.

p38 MAP Kinase Mediates Nitric Oxide-induced Apoptosis of Neural Progenitor Cells

Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome crelease, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.

RNA Interference in Biology and Medicine

First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complexthat degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins—RNAi technology may eventually make that dream a reality.

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin‐linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the α chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a β1 integrin‐dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin‐activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide‐3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor‐induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin‐mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate‐associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.

Amphotericin B Inhibits the Generation of the Scrapie Isoform of the Prion Protein in Infected Cultures

ABSTRACT Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts about the nature of the responsible agent of these diseases remain, it is clear that a protein called PrPSc plays a central role. PrPSc is a conformational variant of PrPC, the normal host protein. Polyene antibiotics such as amphotericin B have been shown to delay the accumulation of PrPSc and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike for Congo red and sulfated polyanions, no effect of amphotericin B has been observed in infected cultures. We show here for the first time that amphotericin B can inhibit PrPSc generation in scrapie-infected GT1-7 and N2a cells. Its activity seems to be related to a modification of the properties of detergent-resistant microdomains. These results provide new insights into the mechanism of action of amphotericin B and confirm the usefulness of infected cultures in the therapeutic research of transmissible spongiform encephalopathies.

PrP-dependent cell adhesion in N2a neuroblastoma cells

The cellular isoform of prion protein (PrPC) is a ubiquitous glycoprotein expressed by most tissues and with a biological function yet to be determined. Here, we have used a neuroblastoma cell model to investigate the involvement of PrP in cell adhesion. Incubation of single cell suspension induced cell–cell adhesion and formation of cell aggregates. Interestingly, cells overexpressing PrP exhibit increased cation‐independent aggregation. Aggregation was reduced after phosphatidylinositol‐specific phospholipase C release of the protein and by pre‐incubation of cells with an antibody raised against the N‐terminal part of PrPC. Our paradigm allows the study of the function of PrP as an intercellular adhesion molecule and a cell surface ligand or receptor.

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.

Involvement of Gadd153 in the pathogenic action of presenilin‐1 mutations

Mutations in the presenilin‐1 (PS1) gene cause early onset familial Alzheimers disease (FAD) by a mechanism believed to involve perturbed endoplasmic reticulum (ER) function and altered proteolytic processing of the amyloid precursor protein. We investigated the molecular mechanisms underlying cell death and ER dysfunction in cultured cells and knock‐in mice expressing FAD PS1 mutations. We report that PS1 mutations cause a marked increase in basal protein levels of the pro‐apoptotic transcription factor Gadd153. PS1 mutations increase Gadd153 protein translation without affecting mRNA levels, while decreasing levels of the anti‐apoptotic protein Bcl‐2. Moreover, an exaggerated Gadd153 response to stress induced by ER stress agents was observed in PS1 mutant cells. Cell death in response to ER stress is enhanced by PS1 mutations, and this endangering effect is attenuated by anti‐sense‐mediated suppression of Gadd153 production. An abnormality in the translational regulation of Gadd153 may sensitize cells to the detrimental effects of ER stress and contribute to the pathogenic actions of PS1 mutations in FAD.